In cattle reproduction, prevalence of artificial insemination in Japan is nearly 100%. However, the conception rate of cattle is decreasing yearly: the first service conception rate of 62.4% and the 1st-3rd service conception rate of 62% in 1989 declined to the first service conception rate of 46.1% and the 1st-3rd service conception rate of 44.6% in 2008. While there are many possible factors contributing to the reduction in the conception rate, one possible factor is considered to be stress, etc., due to the enhanced milk production of cows.
When conception fails, it is necessary to carry out artificial insemination again on the non-gravid female cattle, which imposes a burden on breeding farmers in terms of cost and labor. Thus, there is a need for the enhanced conception rate not only from the viewpoint of female cattle, but from the viewpoint of a bull.
For artificial insemination of livestock, frozen semen aliquoted in a semen straw is usually used. A semen straw for cryopreservation can be prepared by diluting semen in a primary diluent for cryopreservation and then diluting it in a secondary diluent, which is a cryoprotectant-added primary diluent, and filling the liquid in straws, and the semen straw is then freeze-preserved in liquid nitrogen (Non-patent document 1). Conventionally, monolayer straws comprising a single semen preserving layer alone were used, but in recent years, a semen filling device capable of filling two layers was developed (Patent Document 1). This semen filling device can arrange the same cryopreservation solution as that for the semen preserving layer at the cotton plug side, in order to prevent the loss of semen due to contact of the semen preserving layer with the cotton plug.
For semen diluting liquids to be used in freezing semen, ingredients thereof are being improved for the purpose of improving the survival rate and fertilization ability of spermatozoa after freezing and thawing. As the diluent, an egg yolk-based preserving solution comprising egg yolk, saccharides, and a buffer as main ingredients, and a milk-based preserving solution comprising milk as the main ingredients are generally known, and the egg yolk-based preserving solution is widely used on a global basis. Egg yolk has effects of alleviating cold shock, protecting the cell membrane, maintaining the viability of spermatozoa, etc., and these effects are thought to result from lipoprotein and phospholipid in the egg yolk. In order to prevent freezing damage on spermatozoa, various cryopreservatives (Patent Document 2), spermatozoa-activating agents for enhancing the fertility rate (Patent Document 3) and the like are being investigated. During the freezing and thawing process, generally no salts are added to the diluent for freezing, since salts can have adverse effects on spermatozoa.